Human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and the simian immunodeficiency virus (SIV) encode a gene, designated nef (negative effector), that is dispensable for viral replication in vitro (i.e., in tissue culture cells). However, genetic analysis of SIVmac mutants in rhesus macaques has demonstrated that the nef gene is important for maintaining high virus load and for pathogenesis (Kestler et al. 1991). Although numerous studies have been directed at determining the function of the HIV and SIV nef gene in vitro, a critical gap remains in the knowledge of the role of this gene in viral replication and pathogenesis. The goal of this proposal is to elucidate the function of the nef gene both in vitro and in vivo (i.e., a non-human primate model). Recent investigations have revealed that HIV-1 and SIV Nef associates with cellular factors, one of which has protein kinase activity. Accordingly, an important objective within this proposal is to identify these cellular factors and thereby elucidate the mechanisms of HIV and SIV Nef in both human and simian cells. Because SIV is genetically related to HIV and because SIV infection of macaques produces a fatal AIDS-like disease, this highly manipulatable animal model will be utilized to test the in vivo significance of the in vitro studies on nef protein function. HYPOTHESIS The hypothesis is that cellular factors, including a protein kinase, associated with HIV/SIV Nef are critical for Nef function in viral persistence and pathogenesis. SPECIFIC AIM 1: Functional properties of Nef proteins from novel and unique HIV-1 and SIV isolates will be analyzed in vitro. SPECIFIC AIM 2: The mechanism of Nef will be elucidated in in vitro systems; emphasis is directed at characterizing the host cell kinase that associates with this viral protein. SPECIFIC AIM 3: SIVmac mutants in functional domains of Nef will be constructed, characterized in vitro, and analyzed in vivo (i.e., by infection of rhesus macaques) to relate the results of the biochemical analysis of Nef to viral persistence and pathogenesis. SPECIFIC AIM 4: SIV/HIV-1 chimeras (i.e., SHIV) will be constructed by substituting the SIV nef gene with an HIV-1 nef gene, and these chimeric viruses will be tested in rhesus macaques to directly address the function of HIV-1 Nef in vivo. SIGNIFICANCE; The proposed research integrates studies on the function of the nef genes of HIV-1 and SIV. Results of biochemical studies on Nef function of both viruses will be related to in vivo findings in SIV- and SHIV-infected rhesus macaques. A major emphasis of the proposed research is to identify cellular factors which mediate Nef function; accordingly, this knowledge may provide a basis for developing and testing novel anti-viral therapies aimed at inhibiting Nef function and thereby preventing disease progression.